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This is supported by the experiment using singly labeled Fl-Con A prepared by P-PALM. In the titration experiments of Fl-Con A with saccharides, we found that the fluorescence intensity of Fl decreased, similar to the results obtained in AMCA-Fl-Con A (see Supporting Information Figure S-4a). Consistently, the fluorescence anisotropy decreased in the presence of sugar, relative to that in the absence of sugar (see Supporting Information Figure S-5), suggesting that the rather restricted Fl mobility was relaxed by kicking out Fl upon sugar binding. In contrast, the randomly modified Fl-Con A did not show any fluorescence response to the corresponding saccharides (see Supporting Information Figure S-4b), which indicates that tethering of Fl to the proximity of the binding pocket is crucial to this fluorescent sensing. As demonstrated in our previous papers (refs 8a and 8c), the sugar-binding pocket of Con A is rather hydrophobic because of the proximal two Tyr residues and Ile. Similar to the previous DANS-Con A, Fl was kicked out so as to suppress the fluorescence intensity upon the saccharide binding. In addition to this microenvironmental effect, the UV-visible titration experiments (Supporting Information Figure S-6) suggested that the pH change depending on the location of Fl is also an additional factor for the quenching. In the case of Fl, it is well-known that the pH-dependent change takes place between the strong fluorescent form and the weak fluorescent form. By comparing the UV-visible spectral change, it is suggested that the weak fluorescent form slightly increases upon sugar binding in Fl-Con A.
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The darkness difference in the background is mainly due to the released AMCA-Fl-Con A from the cell surface to the bulk solution upon Man-3 addition. The released AMCA-Fl-Con A should bind to Man-3 so that the reddish fluorescence is stronger than that of AMCA-Fl-Con A in the bulk in Figure 6b. Since the background of Figure 6c is more reddish than that of Figure 6b, the darkness seems to be different between them. The difference of the fluorescence spectra of the background was shown by the spectrum mode of CLSM (see Figure S-8).
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It is known that several glucose-dependent pathways generate the fluorescent cofactor NAD(P)H from the nonfluorescent NAD(P). Using this phenomenon, Evans et al. reported the glucose-dependent increase of the fluorescence due to NAD(P)H (so-called the autofluorescence of the cell at 400-500 nm by excitation at 340 nm: Evans, N. D.; Gnudi, L.; Rolinski, O. J.; Birch, D. J. S.; Pickup, J. C. Diab. Technol. Ther. 2003, 5, 807). In our study, such an autofluorescence was not significantly measured under the various glucose concentrations (from 0 to 40 mM, without AMCA-Fl-Con A), confirming that the autofluorescence by the glucose uptake did not influence the present ratiometric fluorescence change of AMCA-El-Con A in the HepG2 cell.
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